Veysel Sabri Hancer

Activation-induced cytidine deaminase mRNA levels in chronic lymphocytic leukemia

The Rai and Binet staging systems, which are used as standard methods for evaluating the prognosis of chronic lymphocytic leukemia (CLL), have some restrictions in identifying patients with early-stage CLL who will progress rapidly. To solve this defect, other prognostic parameters have become important in recent years. Intracellular up-regulation of the AID gene in the leukemic lymphocytes of patients with CLL may be an important parameter for predicting the progression of CLL. In this study, AID mRNA expression levels were evaluated in 50 patients with CLL and 50 healthy controls. AID mRNA expression was significantly higher in patients than in controls. We then evaluated AID mRNA levels according to the stages of CLL. Regarding AID mRNA levels, patients with Rai stages 0, I, and II were compared with patients with stages III and IV, whereas patients with Binet stage A were compared with patients with Binet stages B and C. In patients with higher-risk Rai stages III and IV and Binet stages B and C, activation-induced cytidine deaminase (AID) mRNA levels were also significantly higher. Additionally, we found that the mRNA levels of patients with AID in CLL were eight-fold higher than those in control patients, suggesting that AID overexpression promotes chromosomal abnormalities and is associated with CLL progression and survival. For this reason, and because of the simplicity of quantitative real-time PCR analysis, AID might be a useful clinical parameter after its importance is confirmed in larger and multivariate studies.

The impact of platelet membrane glycoprotein Ib alpha and Ia/IIa polymorphisms on the risk of thrombosis in the antiphospholipid syndrome.

BACKGROUND Pathogenesis of thrombus formation in antiphospholipid syndrome (APS) is not clear. Platelet membrane glycoprotein (GP) receptors play important roles in development of thrombosis. OBJECTIVES We investigated the association between development of thrombosis in APS and polymorphisms of GPIb alpha variable number of tandem repeats (VNTR), Kozak, and GPIa C807T. Patients/Methods Sixty patients with APS (30 with proven thrombosis and 30 without thrombosis) and 63 controls were included. Presence of GPIa C807T polymorphism was determined with real-time PCR and GPIb alpha Kozak and VNTR polymorphisms by conventional PCR. RESULTS Frequency of C807T TT genotype was significantly higher in APS with thrombosis than APS without thrombosis (p=0.023) and also in APS with multiple thrombi compared to APS without thrombi (p=0.023). Frequency of Kozak TC genotype was higher in APS with arterial thrombosis compared to APS with venous thrombosis, controls, and APS without thrombosis (p=0.03, p=0.0007, and p=0.0024 respectively). D allele frequency and D allele carrier state for VNTR were significantly less in APS than controls (p=0.0018 and p=0.0046 respectively). CONCLUSIONS C807T TT genotype may confer a risk for thrombosis and Kozak TC genotype for arterial thrombosis. D allele of VNTR may protect from APS. No patients with C807T TT or Kozak TC genotypes carried the protective DD genotype of VNTR. These polymorphisms may increase risk for both arterial and venous thrombosis. The utility of prophylaxis with anti-platelet drugs in at least a subgroup of APS patients should be investigated with clinical trials.

Polymorphisms of the angiotensin-converting enzyme and angiotensinogen gene in patients with atrial fibrillation

Activation of the renin–angiotensin system (RAS) is associated with atrial fibrillation (AF). The aim of this study was to investigate the relation between AF and polymorphisms in RAS. One hundred and fifty patients with AF, 100 patients with no documented episode of AF and 100 healthy subjects were consecutively recruited into the study. The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism, and the M235T, A-20C, and G-6A polymorphisms of the angiotensinogen gene were genotyped. Patients with AF had significantly lower frequency of II genotype of ACE I/D and higher frequency of angiotensinogen M235T polymorphism T allele and TT genotype and G-6A polymorphism G allele and GG genotype compared with the controls. AF patients had significantly larger left atrium, higher left ventricular mass index (LVMI) and higher frequency of significant valvular pathology. ACE I/D polymorphism II genotype, angiotensinogen M235T polymorphism TT genotype and G allele and GG genotype of angiotensinogen G-6A polymorphism were still independently associated with AF when adjusted for left atrium, LVMI and presence of significant valvular pathology. Genetic predisposition might be underlying the prevalence of acquired AF. Patients with a specific genetic variation in the RAS genes may be more liable to develop AF.

Comparison of KRAS Mutation Tests in Colorectal Cancer Patients

The KRAS pathway and studies evaluating KRAS as a prognostic marker in colorectal cancer are discussed along with advances in KRAS gene mutation testing. Highly sensitive real-time polymerase chain reaction (PCR) methods were developed for this purpose. We examined the applicability of direct sequencing and two real-time PCR methods in the diagnosis of KRAS mutations. We used real-time PCR and direct sequencing-based methods to determine applicability of these KRAS mutation tests in 64 colorectal cancers. The two DNA samples found to be mutation positive by real-time PCR were analyzed again after diluting 100-fold. The results were the same. When we applied the same strategy for the direct sequencing, even a 10-fold dilution did not show the mutations. Therefore, we found that sequencing may not be informative when there are only a few mutant cells in the tumor. KRAS mutation screening on formalin-fixed, paraffin-embedded DNA is very efficient with real-time PCR methods in comparison to direct sequencing. The development and adoption of guidelines for KRAS mutation testing are crucial for success.

MPL W515L/K Mutations in Chronic Myeloproliferative Neoplasms

OBJECTIVE The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. MATERIALS AND METHODS A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction. RESULTS In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET (43.9%, 29/66) and 6 had PMF (54.5%, 6/11). In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times (124 months and 71 months, respectively), had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies. CONCLUSION MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. CONFLICT OF INTEREST None declared.

Glycoprotein Ib-alpha Kozak polymorphism in ischemic stroke.

Abstract Background: Recently, a T/C polymorphism in the Kozak sequence of glycoprotein Ib-alpha (GPIb-alpha) gene at position −5 from the initiator ATG codons, has been identified. The presence of −5C allele increases the surface expression of GPIb-IX–V complex in a gene dosage-dependent manner. It has been suggested that higher receptor levels might increase the adhesiveness of the platelets and confer risk for thrombosis. In this study, we aimed to investigate the association between GPIb-alpha Kozak polymorphism and ischemic stroke. Methods: We prospectively and consecutively recruited 231 patients (118 women and 113 men; mean age: 65±14·2 years) with first ever ischemic stroke admitted to Istanbul Faculty of Medicine Edip Aktin Stroke Unit between April 2007 and June 2009. Demographic features, risk factors, clinical, and etiological subtypes were analyzed. As the control group, 220 unrelated healthy subjects were included. Results: We found that 156 patients had TT, 70 patients had TC, and 5 patients had CC genotype. At least one copy of C allele carriers were overrepresented in the ischemic stroke group (32·5%) compared with controls (23%) [odds ratio (OR): 0·61; 95% confidence interval (CI): 0·40–0·93; P = 0·03]. Among etiologic subtypes, the distribution of C allele carriers was the highest in patients with undetermined etiology (45%) and it was significantly higher than controls (OR: 0·36; 95% CI: 0·20–0·65; P = 0·0008). In other subtypes, there was no association with Kozak −5C allele. Conclusion: In conclusion, these encouraging preliminary results show that GPIb-alpha T/C polymorphism might increase the risk of ischemic stroke, especially in those with undetermined etiology.

Overexpression of Fc mu receptor ( FCMR , TOSO ) gene in chronic lymphocytic leukemia patients

Rai and Binet staging systems that have been used as a standard method for evaluating the prognosis of chronic lymphocytic leukemia (CLL) have some restrictions in distinguishing the early stage CLL patients that will progress rapidly. To solve this shortcoming, prognostic parameters other than staging have become important in the recent years. Intracellular upregulation of Fc mu receptor (FCMR, FAIM3/TOSO) gene in the leukemic lymphocytes of the patients with CLL may be an important parameter in predicting the progression of the disease. In this study, FCMR mRNA expression levels were evaluated in 50 CLL patients and in 50 healthy controls. FCMR mRNA expression was found to be significantly higher in CLL patients than in healthy controls. We, then, evaluated FCMR mRNA levels according to the stages of CLL. Rai stage 0, I, II cases were compared with stage III and IV, and Binet A was compared with Binet B and C according to FCMR mRNA levels. In cases with higher risks, Rai stage III, IV and Binet stage B and C, FCMR mRNA levels were also significantly higher. In addition, overexpression of the FCMR seems to be promoting the chromosomal abnormalities. As a result, we found that the mRNA levels of FCMR in the CLL patients are 23-fold higher than that of the control group and this may suggest that it can be associated with the disease progression and survival. For this reason and because of the simplicity of analyzing with Q-PCR, it can be a useful clinical parameter, after its importance has been shown in larger and multi-variate studies.

Prophylactic Eculizumab Use in Kidney Transplantation: A Review of the Literature and Report of a Case with Atypical Hemolytic Uremic Syndrome.

BACKGROUND Atypical hemolytic uremic syndrome (aHUS) is a very rare disease, which presents with microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. Progression to end-stage renal disease (ESRD) from acute kidney injury is observed in 60% of aHUS cases. The prognosis of aHUS patients who undergo kidney transplantation (Ktx) is generally poor, but these patients should be treated prophylactically with eculizumab to prevent recurrence after transplantation. CASE REPORT An 18-year-old man was referred to our center with a history of rapid progression to ESRD with unknown etiology. He had anemia, thrombocytopenia, high levels of LDH, and indirect bilirubin and creatinine on initial laboratory results. Our diagnosis was aHUS due to initial results, normal level of ADAMTS activity, and lack of predisposing factors seen in typical HUS. We planned to perform genetic analysis for the patient and the donor candidate (mother). The variations found on exon 7 of the CFH gene had not been reported previously. According to PolyPhen analysis, this mutation was reported as a potential cause for aHUS. We decided to perform Ktx under eculizumab prophylaxis. Weekly administration of prophylaxis was extended to 1 month. The graft functioned immediately after Ktx. The patient has completed his first year uneventfully in our follow-up, with a creatinine 0.79 mg/dl at his last control visit. CONCLUSIONS We found favorable results of an aHUS case successfully treated with kidney transplantation combined with short-term prophylactic eculizumab therapy.

The Relationship between P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome: A Pilot Case-Control Study.

Objective: The selectins are cell adhesion molecules that mediate the interactions among leukocytes, activated platelets, and endothelial cells. We aimed to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with antiphospholipid syndrome (APS). Materials and Methods: The diagnosis and classification of APS were based on the report of an international workshop. Genomic DNA was extracted from citrated blood samples of all subjects. Three single nucleotide polymorphisms associated with the P-selectin coding region (S290N, c.1087G>A; N562D, c.1902G>A; T715P, c.2363A>C) were assessed. Results: There were 26 APS (65%) patients with thrombosis. The number of patients without thrombosis was 14 (35%). The frequency of the N562D-DN genotype was significantly higher in patients with APS than in healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared with patients with no thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). Conclusion: Our results suggest that the N562D polymorphism of the DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS.

Chronic lymphocytic leukemia developing in a patient with Janus kinase 2 V617F mutation positive myeloproliferative neoplasm

Dear Editor, The Janus kinase 2 (JAK2) V617F mutation is found to be present with different frequencies in patients with myeloproliferative neoplasms (MPNs). JAK2 mutation has been reported as absent in chronic lymphocytic leukemia (CLL) patients. The coexistence of CLL and MPN is a rare entity. The coexistence of these two clonal hematological disorders in a patient raises the possibility that both are derived from the same pluripotent stem cell, or they are coincidental. Here, we present a patient who had both JAK2 positive MPN and CLL simultanenously. A 56-year-old male patient was admitted to our hematology department with leukocytosis and thrombocytosis in February 1999. The physical examination was unremarkable. The hemoglobin level was 13.6 g/dL; the hematocrit was 41.8%; the platelets were 985×10/L; and white blood cells were 25.1×10/L. The peripheral blood smear revealed 3% bands, 43% neutrophils, 2% eosinophils, 48% lymphocytes, and 4% monocytes. The patient had lymphocytosis with an absolute number of 10.08×10/L, and the peripheral blood flow-cytometry revealed CD5 (99.9%), CD19 (82.9%), CD5 +19 (82.3%), CD20 (86.9%), and CD23 (38.8%) positivities supporting the diagnosis of CLL. Abdomen ultrasonography showed splenomegaly. Due to the presence of thrombocytosis, a bone marrow aspiration and biopsy was done showing hypercellular bone marrow, erythroid hyperplasia with normoblastic maturation, normal myeloid maturation, increased megakaryocytes with dysplastic features, and grade II fibrosis with a lymphocyte percentage of 9%. Another bone marrow aspiration and biopsy was performed in May 1999 which showed 40% of lymphocytes. The conventional cytogenetic study showed no chromosomal abnormalities, and the BCR-ABL was negative. Del17p, del11q, and del13q were negative by fluorescence in situ hybridization. The diagnoses of the patient were Philadelphia (Ph)negative MPN and CLL. Splenomegaly was thought to be related to MPN, so CLL was staged as Rai 0. The patient did not have treatment indication for CLL, so hydroxyurea (HU) and acetylsalicylic acid (ASA) treatment was initiated in March 1999. While on HU therapy, the patient developed leg ulcers, so HU was discontinued in April 2007. His hemoglobin level was 9.4 g/dL, and there were no signs of hemolysis, so chlorambucil (CB) was started. During the follow-up, he had an acute abdomen which resulted in resection of a segment of jejunum and ileum due to a transmural infarction in January 2008. CB was discontinued, and HU was restarted in March 2008. He is still on HU therapy 2 g/ day with ASA 100 mg/daily. The JAK2 V617F point mutation was detected as positive, and the analysis was carried out as previously described [1]. Lymphocytes were separated from the heparinized venous blood by density gradient centrifugation, and the mutation was found to be positive in A. E. Eskazan (*) :A. Salihoglu : T. Soysal Division of Hematology, Department of Internal Medicine, Cerrahpasa Faculty of Medicine, Istanbul University, Kocamustafapasa, Fatih, Istanbul, Turkey e-mail: emreeskazan@hotmail.com